Triple in WD-repeat proteins. While the protein sequence provides

            Triple A Syndrome
is an inherited autosomal recessive genetic disorder defined by three features:
adrenal insufficiency, alacrima (absence of tear secretion), and achalasia (inability
of the lower esophageal sphincter at the cardia to relax). Of these features,
alacrima is often the first to develop, followed by one or both of the
remaining characteristics. While these hallmark features define triple A syndrome,
researchers have shown that the disease can cause autonomic dysfunction. Since
this division of the nervous system controls diverse involuntary processes such
as blood pressure and body temperature, this disease is highly variable in its
severity, age of onset, and number of symptoms shown. Triple A Syndrome is also
associated with other neurological impairments such as intellectual disability,
speech impairment, and microcephaly. Furthermore, affected individuals also commonly
experience muscle weakness and movement problems. As the condition is a
progressive disorder, many of the neurological symptoms of triple A syndrome
may present later in life and worsen over time. Currently, there is no cure for
triple A syndrome and available treatments are tailored to manage the
individual signs and symptoms of the disease.

            In an
attempt to find the genetic cause of triple A syndrome, Huebner et. al. investigated 47 affected
families using a genome-wide systematic scan and identified the AAAS gene located on chromosome 12q13.

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Sequence analysis showed that the gene contains 16 exons and encodes a protein
of 546 amino acids with an estimated molecular mass of 60 kDa. This protein, referred
to as ALADIN (alacrima achalasia adrenal insufficiency
neurologic disorder protein) also contains four WD repeats. This is
interesting because WD-repeats are known to form b-propeller structures involved in protein-protein interactions.

Additionally, several other diseases such as Cockayne syndrome and dactylaplasia
have been linked to mutations in WD-repeat proteins. While the protein sequence
provides clues on how this protein could function in normal cells, it would be
premature to conclude the activities of ALADIN based on the protein sequence
data alone since WD-repeat proteins are involved in diverse processes such as
signal transduction, RNA processing, and vesicular trafficking. Thus, after
ALADIN was identified, researchers next looked to determine its tissue specific
expression and its subcellular localization to better understand how defects in
the protein are involved in triple A pathogenesis.

            Thirteen
nonsense, ten frameshift and five aberrant splicing mutations in the AAAS gene have been found in patients
with triple A syndrome and are predicted to encode truncated versions of ALADIN
lacking most or all of the C-terminus. Other disease associated mutations have
been found that affect the WD-repeat regions or the 15th amino acid (Q15).

Cho et. al. first determined the expression
levels of wild-type ALADIN in human tissues using the multiple human tissue
northern (MTN) blot technique. Labelled DNA probes consisting of exon 1 or
spanning exons 4-16 of the gene were used to detect AAAS mRNA in 16 different
human tissues. The MTN blot results showed that the gene was expressed in all
tissues tested, however the gene was more highly expressed in the placenta, testis,
pancreas, adrenal gland, pituitary gland, kidney, various gastrointestinal
structures, and the cerebellum. After this, the authors looked to see if the
AAAS mRNA was transcribed in all tissues. However, in this study, expression of
ALADIN was only probed in human adrenal gland, pancreas, pituitary, kidney, and
placental samples due to limited tissue availability. ALADIN levels were probed
by western blot analysis using the anti-CNE19 antibody specific for ALADIN. Results
showed that the protein was expressed in pancreas, adrenal and pituitary glands
but not in the kidney, skeletal muscle and placenta. Specific expression of
ALADIN in these tissues may explain why disruption of the protein results in
the specific triple A phenotype.    

After it was shown that AAAS is ubiquitously transcribed but
only transcribed in select tissues, the subcellular localization of wild type
and mutant ALADIN proteins were investigated to elucidate the normal function
of the protein and its role in triple A syndrome. Previous cell fractionation
assays had demonstrated that ALADIN is found in the nuclear membrane fraction.

To determine if and how the protein is associated with the nuclear pore
complexes (NPC’s), Cronshaw and Matunis first examined the subcellular
localization of the wild-type protein by transfecting HeLa cells with
GFP-ALADIN. HeLa cells were then fixed and labelled with antibodies against
Nup358 and Tpr to visualize individual NPCs. Using deconvoluted microscopy,
they observed that the NPC and ALADIN fluorescence signals co-localized. They
also noted that the ALADIN and Nup358 signals overlapped more closely than the
ALADIN and Tpr signals, suggesting that ALADIN is found at the cytoplasmic face
of the NPC. These results implicate ALADIN as a nucleoporin.

            Next,
Cronshaw and Matunis examined the specific domains of the protein essential to
target ALADIN to the NPC.  As many of the
triple A mutations result in the C-terminal truncation of ALADIN, the
investigators examined the subcellular localization of ALADINR478X, the
most severely truncated mutant. HeLa cells were transfected with GFP- ALADINR478X
and the NPCs were visualized as before (with antibodies against Nup358 and
Tpr). While wild-type ALADIN had been shown to localize to NPCs, the absence or
truncation of the C-terminus abolished NPC localization and resulted in a diffuse
cytoplasmic localization. This result suggested that the C-terminus of ALADIN
alone may be sufficient to target the protein to NPCs. However, when HeLa cells
were transfected with the C-terminal domain of the protein (GFP- ALADIN317-546),
the fragment was shown to localize to the cytoplasm.

The authors then examined the N-terminal domain to see if
was essential for NPC targeting. When transfected into HeLa cells, a GFP-tagged
ALADIN mutant lacking the first 100 residues was found to be distributed
throughout the cell, indicating that the N-terminal domain is also needed to
target ALADIN to the NPC. However, since the N-terminal and C-terminal
truncated proteins have different subcellular localizations, these two termini
must play different roles in the targeting process. Since the N-terminal
truncations are found throughout the cell, including the nucleus, it may
contain a signal responsible for cytoplasmic retention, however there is not
strong evidence to support this claim.

            After this,
the authors examined the consequences of point mutations in the WD-repeat domains
of ALADIN. As WD-repeats form a b-propeller structure, mutations in these domains may disrupt
proper protein folding and result in failure of ALADIN to localize to NPCs.

Consequently, GFP-ALADINH160R, GFP-ALADINS263P, and
GFP-ALADINV313A are mislocalized to the cytoplasm. However, one point
mutation, Q15K, did not disrupt the NPC localization of ALADIN. As these
WD-repeats are known to be involved in protein-protein interactions, this
mutation may disrupt the ability of ALADIN to interact with one or more
proteins or to form a protein complex essential for its function. Indeed, there
are rare cases of triple A that are not associated with mutations in AAAS, suggesting that interacting
protein partners or some other unknown mechanism may also be responsible for
disease.

            Finally,
the authors investigated if failure of ALADIN to localize to the NPC disrupts
general nucleocytoplasmic transport or NE/NPC structure/assembly. Using a human
fibroblast cell line derived from a patient with triple A syndrome and
containing a splice-site mutation resulting in the production of nonfunctional
ALADIN proteins, Cronshaw and Matunis examined the structure of the NPCs and NE
via electron microscopy. Compared with a normal fibroblast cell line, the nuclei,
NEs, and NPCs displayed a normal morphology. These results were confirmed through
immunofluorescence microscopy using nucleoporin specific antibodies. In these
experiments, cells were also immunostained with antibodies against importin a and
b,
which also displayed normal localization compared to controls. Thus, ALADIN
mutations result in functional rather than structural defects. This is
consistent with the severity of triple A syndrome, as disease itself is not
lethal and most tissues are unaffected, signifying that NPCs are intact and
essential NPC functions are retained.

As stated previously, there are some rare
cases of triple A syndrome that are not associated with mutations in AAAS,
which suggests that other modifying genes/factors must play a role in pathogenesis.

NDC1 is a transmembrane nucleoporin involved in NPC assembly. Loss of this
nucleoporin is highly deleterious as localization of FG-repeat containing
nucleoporins is impaired. While identifying NDC1-associated proteins, Yamazumi
et. al. demonstrated that ALADIN and NDC1 interacted with each other. 293T
cells were transfected with FLAG-tagged NDC1 and lysates were
immunoprecipitated with an anti-FLAG antibody. Co-immunoprecipitated proteins
were identified by LC-based tandem mass spectrometry (MS/MS) showing that
ALADIN is indeed co-precipitated with NDC1. This result was confirmed through
affinity chromatography using GST, a GST-NDC1 fusion protein, and in vitro
translated ALADIN.  

To determine if endogenous NDC1 interacts
with ALADIN in vivo, lysates from HeLa cells were subjected to
immunoprecipitation with an antibody against NDC1 or ALADIN. Immunoblotting
results showed that both of the proteins were able to “pull down” the other and
confirmed an interaction in vivo. Indeed, in HeLa cells transfected with
FLAG-NDC1 and GFP-ALADIN, the two fusion proteins were seen to co-localize at
the nuclear rim via confocal microscopy. These sets of experiments show that
NDC1 and ALADIN bind to each other and are associated at the NE in living
cells.

Since failure of ALADIN to localize to the
NPC is known to at least partially cause the triple A phenotype, the authors
investigated whether the presence of NDC1 is necessary to target ALADIN to the
NPCs. HeLa cells were treated with two different shRNA against NDC1, both of
which were shown to nearly completely block expression of NDC1 protein via
immunoblotting. To determine how the absence of NDC1 affected ALADIN
localization, HeLa cells were then transfected with GFP-ALADIN and one of the
shRNA’s against NDC1 or control shRNA. Confocal imaging revealed that in the
control co-transfected cells, GFP-ALADIN was found to localize to the NPCs.

However, in cells co-transfected with shRNA against NDC1, GFP-ALADIN was found
to be dispersed in the cytoplasm. These results show that NDC1 is important in
ALADIN localization to the NPCs and may act to tether ALADIN at the cytoplasmic
face of the NPC. These results also suggest that the NDC1 gene may be mutated
in triple A syndrome patients without mutations in AAAS gene.

Since loss of NDC1 was shown to be highly
deleterious as localization of FG-repeat containing nucleoporins is impaired,
the authors lastly looked at how loss of NDC1 affected nucleocytoplasmic
import. This was especially of interest since previous research showed that
ALADIN is involved in the nuclear import of proteins containing the NLS of
SIV40 large T antigen, but not the import of proteins containing other nuclear
localization signals. The authors tested if the nuclear import of a
Dronpa-tagged SV40 NLS sequence or Dronpa-tagged XRCC1(containing a different
NLS) was impacted in HeLa cells co-transfected with shRNA against NDC1. Imaging
results showed that was Dronpa-NLSSV40 mislocalized to the cytoplasm in cells
transfected with shRNA-NDC1 while Dronpa-XRCC1 still localized to the nucleus.

This shows that NDC1 is required for selective nuclear import of NLSSV40 and
may indicate that NDC1-mediated anchoring of ALADIN to NPCs is essential for import
of essential proteins whose absence in the nucleus contribute to the triple A
phenotype.

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